Central to the ability of vascular cells to adhere to the extracellular matrix and to each other is an abundant supply of cell surface adhesion molecules that, in addition to influencing the adhesive phenotype of the cell, are also capable of transmitting signals into, and responding to signals from, the cell interior. Such post-ligand events occur by virtue of the ability of these transmembrane proteins to interact with intracellular kinases and phosphatases, G-proteins, adapter proteins, and cytoskeletal components. PECAM-1, (also known as CD31) is a 130 kDa member of the Immunoglobulin superfamily that is expressed on the surface of circulating platelets, monocytes, neutrophils, and selected T-cell subsets. It is also a major constituent of the endothelial cell intercellular junction, where up to one million PECAM-1 molecules are concentrated. The overall goal of this competitive renewal application is to build on recent progress in the field of PECAM-1 biology, and to continue to explore the hypothesis that PECAM-1, owing to its unique distribution on platelets, leukocytes, and endothelial cells, lies at the nexus of thrombosis and inflammation. There is growing appreciation in the fields of thrombosis, hemostasis, and vascular biology that these two pathophysiological conditions are inextricably and mechanistically linked. PECAM-1, via its ability to inhibit platelet activation, suppress cytokine production and responsiveness, stimulate vessel wall production of prostacyclin, and support the integrity of endothelial cell-cell junctions, appears to play a significant role in each of these interrelated processes. In the coming grant period, we propose to (1) Determine the functional domains of PECAM-1 that are required for its support of endothelial cell barrier function, (2) Identify downstream effectors of PECAM-1 that enable it to regulate cytokine production and cytokine responsiveness in leukocytes exposed to inflammatory stimuli, (3) Characterize the naturally-occurring ligands for PECAM-1 that evoke endothelial cell secretion of prostacyclin, and the functional domains of the receptor that are required to support generation of this endothelial-derived anti-thrombotic and anti-inflammatory agent, and (4) Establish strains of temporally-regulated, tissue-specific transgenic mouse lines expressing selected PECAM-1 variants that lack specific functional domains. Together, these studies comprise a coordinated, focused research program designed to improve our understanding of the function of this novel vascular cell adhesion and signaling molecule in the blood and vascular cells in which it is expressed. We expect that information derived from this investigation will lead to improved understanding of the molecules and events that regulate inflammation, thrombosis, and the immune response. PUBLIC HEALTH RELEVANCE: There is growing recognition that thrombosis, cardiovascular disease, and inflammation are inextricably linked processes that influence each other in ways that are still not well understood. PECAM-1 is a 130 kDa member of the Immunoglobulin superfamily that functions as an inhibitory receptor on platelets and leukocytes, and is also a prominent component of endothelial cell intercellular junctions. The goal of this proposal is to improve our understanding of the function of this novel vascular cell adhesion and signaling molecule in the blood and vascular cells in which it is expressed.